Interleukin-6 is released into the plasma upon injury or infection by different cell typs. It is involved in a spectrum of activities like immune defense, hematopoiesis, maturation of megakaryocytes, platelet production and acute phase response (1).
Besides playing a central role in host defense, IL-6 is involved in the pathogenesis of a variety of diseases like plasmocytoma/myeloma, osteoporosis and neoplastic and autoimmune diseases (1).
The IL-6 receptor complex on target cells consists of two different subunits, an 80-kDa specific ligand binding subunit (IL-6Ra) and a 130-kDa signal-transducing protein (gp130) (2-4). IL-6 binds to the IL-6Ra and the complex of IL-6/IL-6Ra becomes associated with a dimer of gp130, thereby initiating the IL-6 signal. IL-6 by itself has no measurable affinity to gp130 (5,6).
Interleukin-6 is a protein characterised by N-terminal heterogeneity. It has been reported (7) as a 184 amino acids (this amino acids numbering will be followed in this patent application). Secondary structure predictions and protein modeling pointed out that IL-6 is a member of the hematopoietic cytokine family characterized by four antiparallel a-helices (A, B, C, and D) (8,9). LIF (leukemia ihibitory factor), CNTF (ciliary neurotrophic factor), IL-11, CT-1 (cardiotrophin-1) and OSM (oncostatin M) also belongs to this family. They all use the gp130 protein in their receptor complex, which explains their overlaping bioactivities (1, 10, 11).
Deletion studies of IL-6 showed that the N-terminal 28 amino acid residuces are dispensable for the biological activity of this molecule. Removal of more than 28 amino acids inactived the protein (12). Further studies predicted that the C-terminus and the end of the A-B loop/beginning of the B-helix (region 2c, residues G77-E95) are involved in the interaction with the IL-6R.alpha. (9, 13-16). These results were corroborated by the recently published human IL-6 model (9) where these two regions were in close proximity.
At present, two interaction sites of IL-6 with gp130 are identified.
i. Epitope mapping of the IL-6 protein with neutralizing mAbs provided evidence that the residues Q152-T162 (beginning of the D-helix) are involved in gp130 interaction (17, 18). Analysis of chimeric human/mouse IL-6 proteins revealed the presence of an epitope within the beginning of the A-B loop of IL-6 which was involved in contacting and activating gp130 (9, 19). Recently, this result was confirmed by demonstrating that leucine 57 is invoved in this interaction (20). This region is in close proximity of the beginning of helix D leading to the assumption that these two regions together form a common interaction site with one gp130 (9, 19, 21). PA0 ii A second interaction site with gp130 was definded in analogy to the GH (growth hormone)/GHR.sub.2 complex, the structure of which was solved by X-ray analysis (22). It was speculated, that the parts of the GH important for the interaction with the second GHR are the same in the IL-6 protein important for interaction with one gp130 (23, 24). Indeed, the substitution of two amino acids in the A-helix (Y31D/G35F) and two amino acids in the C-helix (S118R/V121D) also lead to an IL-6 mutant protein with nearly normal affinity to the IL-6Ra, but no bioactivity. These four amino acids seem to be important for the interaction with a second gp130 protein (24, 25).
In view of the previously discussed IL-6 involvement in the pathogenesis of some diseases, the development of inhibitors of IL-6 activity has therefore been the subject of active research. For this purpose, different approaches have been pursued, including the use of antibodies against IL-6, gp130 or gp80; the use of soluble gp130; or the use of muteins for IL-6, or IL-6 Receptor.
The Applicant has investigated the possibility of synthesising new IL-6 muteins that can act as IL-6 receptor antagonists. With this aim, one scientific approach to follow is to synthesise muteins that retain the ability to bind IL-6R.alpha., but have lost the capacity to recruit gp130. Therefore, the optimal molecule should be the one that does not show IL-6 activity but shows a higher IL-6R.alpha. binding than IL-6 and that contains as few mutations as possible with respect to IL-6, in order to reduce the risks of antigenicity.